US11052158B2 - Delivery of urea to cells of the macula and retina using liposome constructs - Google Patents

Delivery of urea to cells of the macula and retina using liposome constructs Download PDF

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US11052158B2
US11052158B2 US16/326,195 US201716326195A US11052158B2 US 11052158 B2 US11052158 B2 US 11052158B2 US 201716326195 A US201716326195 A US 201716326195A US 11052158 B2 US11052158 B2 US 11052158B2
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Troy Bremer
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6917—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a lipoprotein vesicle, e.g. HDL or LDL proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • the eye is a very active organ with a constant high-volume circulation of blood and other fluids in and around the globe.
  • the retina is a layer of nerves that lines the back of the eye and contains specialized photoreceptor cells, called rods and cones, which sense light.
  • the retina sends light signals to the visual cortex of the brain through the optic nerve. Cone cells are most concentrated in a small area of the retina called the macula.
  • the choroid is a highly vascular structure between the retina and the white outer layer of the eye, the sclera. The choroid acts as both a source of oxygen and nutrients to the retina, as well as a drainage system of the aqueous humor from the anterior chamber.
  • the eye is filled with a gel-like substance called the vitreous or vitreous body.
  • the vitreous body is an orb-shaped structure of mostly water with a significant concentration of hyaluronan and collagen, plus lesser amounts of a variety of other proteins.
  • the posterior portion of the vitreous body is in direct contact with the retina. Networks of fibrillar strands extend from the retina and insert into the vitreous body to attach it to the retina. See Sebag, Graefe's Arch. Clin. Exp. Ophthalmol. 225:89-93 (1987).
  • the standard administration of currently approved drugs for pathologies of the retina is intra-vitreal injection of a 100 microliter dose using a 26-30 gauge needle, delivered through a structure in the middle layer of the eye, the pars plana, and released in the central portion of the vitreous. It has been established by pharmacokinetic analysis that drugs injected into the vitreous dissipate within a few hours to outer tissues of the eye and are totally removed after 24 hours.
  • the typical 100 microliter injection is diluted by a factor of 50 to 1 before a small concentration moves to the area of prime interest, the macula.
  • ophthalmic drug formulations that can deliver a therapeutically effective dose, particularly of a highly water-soluble active agent such as urea, to the back of the eye over an extended period for the treatment of chronic diseases, such as, for example, diabetic retinopathy.
  • This disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a liposome construct and a pharmaceutically acceptable carrier
  • the liposome construct comprises an agglomerate of small unilamellar vesicles (SUVs), wherein the SUVs comprise urea encapsulated within the SUVs, wherein the SUVs have a specific gravity that is greater than about 1.05, a z-average diameter of less than about 220 nm, and a polydispersity index value (PdI) of less than about 0.30.
  • the z-average diameter is less than about 200 nm.
  • the pharmaceutically acceptable carrier can optionally comprise urea.
  • the pharmaceutical composition is in the form of an emulsion or a suspension.
  • the SUVs have a lipid bilayer (i.e., a lamella) that surrounds a central compartment.
  • the lamella comprises one or more phospholipids and no cholesterol.
  • the lamella comprises (i) one or more phospholipids and (ii) less than about 70 mol % cholesterol, or 1-9 mol % cholesterol, or 34-69 mol % cholesterol, or 42-69 mol % cholesterol, or 10-20 mol % cholesterol, or 20-30 mol % cholesterol, or 30-40 mol % cholesterol, or 40-50 mol % cholesterol, or 50-60 mol % cholesterol, or 60-69 mol % cholesterol.
  • the lamella comprises one or more of cholesterol, dioleoyl phosphatidylcholine (DOPC), dioleyl phosphatidylethanolamine (DOPE), dioleoyl trimethylammonium propane (DOTAP), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylcholine (DSPC), phosphatidylcholine (PC), and palmitoyl oleoyl phosphatidylcholine (POPC).
  • DOPC dioleoyl phosphatidylcholine
  • DOPE dioleyl phosphatidylethanolamine
  • DOTAP dioleoyl trimethylammonium propane
  • DPPC dipalmitoyl phosphatidylcholine
  • DPPG dipalmitoyl phosphatidylglycerol
  • DSPC distearoyl phosphat
  • the lamella consists essentially of 58 mol % DPPC and 42 mol % cholesterol; 58 mol % DOPC and 42 mol % cholesterol; 58 mol % POPC and 42 mol % cholesterol; 29 mol % DPPC, 42 mol % cholesterol, and 29 mol % DPPG; 80 mol % POPC and 20 mol % DOTAP; 67 mol % DMPC and 33 mol % DMPG; or 33 mol % DPPC, 13 mol % DSPC, 32 mol % DOPC, 17 mol % 18:2 PC, 5 mol % 20:4 PC.
  • the lamella consists essentially of 58 mol % DOPC and 42 mol % cholesterol.
  • the SUVs comprise a surface modifying group such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the SUVs can have an encapsulation efficiency of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, the SUVs have an encapsulation efficiency of at least about 20%.
  • a packed pellet of the SUVs comprises at least about 0.1 mg, and preferably at least about 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, or 0.5 mg of encapsulated urea per microliter of packed pellet.
  • the amount of encapsulated urea will depend upon the desired dosage for delivery.
  • One embodiment is directed to a method of inducing posterior vitreous detachment (PVD) in a subject having or susceptible to disease or disorder of the eye that can be treated or prevented by inducing PVD, the method comprising administering to the vitreous of the subject a pharmaceutical composition comprising the liposome construct of the invention.
  • the disease or disorder can be, for example, diabetic retinopathy or vitreomacular adhesion (VMA).
  • a method of treating diabetic retinopathy or VMA in a subject comprising administering to the vitreous of the subject a pharmaceutical composition of the invention.
  • the methods of the invention can comprise administration of the pharmaceutical composition of the invention by intravitreal injection.
  • intravitreal injection is through the pars plana.
  • the methods of the invention can comprise administration wherein the subject is in a supine position.
  • Specific embodiments provide release characteristics of the liposome constructs. In some aspects, at least 80% of the urea is released from the liposome construct within 24 hours after administration. In some aspects, at least 80% of the urea is released from the liposome construct within 8 hours after administration. In some aspects, at least 80% of the urea is released from the liposome construct within 4 hours after administration.
  • Embodiments of the invention include the use of a liposome construct or composition of the invention to induce posterior vitreous detachment (PVD) or to treat or prevent a disease or disorder of the eye that can be treated or prevented by inducing PVD.
  • a liposome construct or composition of the invention to induce posterior vitreous detachment (PVD) or to treat or prevent a disease or disorder of the eye that can be treated or prevented by inducing PVD.
  • One embodiment of the invention includes the use of a pharmaceutical composition comprising a liposome construct of the invention to treat or prevent diabetic retinopathy or vitreomacular adhesion (VMA).
  • VMA diabetic retinopathy or vitreomacular adhesion
  • kits comprising a liposome construct or pharmaceutical composition of the invention.
  • FIGS. 1A-1E show carboxyfluorescein leakage over a 24-hour period from intact and lysed liposome constructs made from Formulation 1 (as disclosed herein) ( FIG. 1A ), Formulation 3 (as disclosed herein) ( FIG. 1B ), Formulation 8 (as disclosed herein) ( FIG. 1C ), Formulation 11 (as disclosed herein) ( FIG. 1D ), or Formulation 12 (as disclosed herein) ( FIG. 1E ).
  • Formulations are described in Table 1.
  • FIG. 2A-2D show stability over a 24-hour period of urea-encapsulated liposome constructs in 1 ⁇ PBS ( FIG. 2A, 2B ) or rabbit vitreous humor ( FIG. 2C, 2D ).
  • Formulations #1, #2, #3, #4, and #5 in the graphs correspond, respectively, to Formulations 1, 2, 3, 12, and 14 described in Table 1.
  • FIG. 3A-3C show stability over a 7-day period of urea-encapsulated liposome constructs made from Formulation 2 (described in Table 1) and stored at 4° C. ( FIG. 3A ), room temperature ( FIG. 3B ), or 37° C. ( FIG. 3C ).
  • FIG. 4 shows a flow chart of a reverse-phase evaporation method for production of liposome constructs comprising encapsulated urea.
  • FIG. 5 shows in vitro urea release data for four batches of liposome constructs.
  • FIG. 6A-6B show the relationship between the amount of encapsulated urea in a 100 ⁇ L dose (40% liposome vol./60% urea buffer vol.) and the concentration of DOPC ( FIG. 6A ) or cholesterol ( FIG. 6B ).
  • FIG. 7A-7B show representative fundus photographs of the Group 2a animal immediately post dose (Day 0), and on the indicated timepoints thereafter.
  • the left eye (OS) received an intravitreal injection of balanced salt solution; the right eye (OD) received an intravitreal injection of 96 mg urea in solution.
  • Hazy appearance of vasculature in the right eye is due to the presence of the drug product.
  • Arrows in OD panels on days 4, 7, and 14 indicate drug product.
  • FIG. 8A-8B show representative optical coherence tomography (OCT) images of the Group 2b animal at the indicated post-dose timepoints.
  • OCT optical coherence tomography
  • the corresponding fundus image is shown to the left of each sub-panel, with a green line indicating the position of the OCT image.
  • the left eye (OS) received an intravitreal injection of balanced salt solution; the right eye (OD) received an intravitreal injection of 192 mg urea in solution.
  • FIG. 9 shows representative fundus photographs of Group 3 (25 mg urea), Group 4 (50 mg urea), and Group 5 (2.5 mg urea) animals at 1 and 35 days after OD injection of urea solution.
  • FIG. 10A-10B show representative OCT images of the Group 3 (25 mg urea) ( FIG. 10A ) and Group 4 (50 mg urea) ( FIG. 10B ) animals at the timepoints indicated.
  • the corresponding fundus image is shown to the left of each sub-panel.
  • Some instances of PVD are indicated by arrows.
  • FIG. 11A-11B show representative B-scan images of Group 3 (25 mg urea) ( FIG. 11A ) and Group 4 (50 mg urea) ( FIG. 11B ) animals before and 35 days after OD injection of urea solution.
  • FIG. 12A-12B show Day 32 electroretinographs.
  • FIG. 12A shows graphs of dark-adapted control (upper panels) and Group 8 (lower panels) animals exposed to blue light.
  • FIG. 12B shows graphs of dark-adapted control (upper panels) and Group 8 (lower panels) animals exposed to red light.
  • Embodiments of the present invention provide novel formulations for delivery of urea to the retina and macula.
  • the liposome constructs of the embodiments of the invention can selectively and specifically release urea at the target area within the eye to treat, prevent, diagnose, and/or monitor a disease or disorder of the eye.
  • Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation, and nucleic acid sequences are written left to right in 5′ to 3′ orientation. Amino acids are referred to by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.
  • a “liposome” is a spherical vesicle with a lipid bilayer surrounding a central compartment. Liposomes can be classified on the basis of the structure of the lipid bilayer. Unilamellar vesicles have one bilayer surrounding the central compartment, while multilamellar vesicles (MLVs) have more than one bilayer surrounding the central compartment.
  • MLVs multilamellar vesicles
  • Liposomes can also be classified on the basis of size: small unilamellar vesicles (SUVs) are typically about 20-100 nm in diameter; large unilamellar vesicles (LUVs) are typically greater than 100 nm in diameter; and giant unilamellar vesicles (GUVs) are typically greater than about 250 nm in diameter. MLVs are typically about 100-500 nm in diameter.
  • the “percent encapsulated” is 100 ⁇ [(amount of active agent encapsulated)/(total amount of active agent)].
  • a “liposome construct” is a particle comprising an agglomerate of SUVs.
  • the liposome constructs of the invention are individual SUVs that self-assemble into liposome constructs, which are denser than the vitreous of the eye. Without wishing to be bound by theory, the individual SUVs are held together by intermolecular forces characterized by charge-sharing. Unexpectedly, the liposome constructs maintain their globular, gel-like structure once administered, such that they do not disperse or break apart during delivery throughout the vitreous body, but instead can sink through the vitreous and blanket the retina.
  • the liposome constructs can comprise an emulsifier or binding agent to enhance agglomeration.
  • the liposome constructs can comprise a surface group (for example, PEG) that facilitates direct or indirect secondary binding between SUVs, which can be hydrolyzed under certain conditions, such as a change in pH.
  • Particle size can be expressed as a “z-average diameter,” which is the mean diameter based upon the intensity of scattered light.
  • the “polydispersity index (PdI)” is an estimate of the width of the particle size distribution.
  • Particle size distribution in a sample can also be expressed in “D-values,” which are based on percentage mass of particles in the sample.
  • the “D90” is the diameter at which 90% of a sample's mass is comprised of smaller particles.
  • the “D50” is the diameter at which 50% of a sample's mass is comprised of smaller particles.
  • the “D10” is the diameter at which 10% of a sample's mass is comprised of smaller particles.
  • the terms “vitreous,” “vitreous body,” “vitreous humor,” and “vitreal fluid,” are used interchangeably to refer to the gelatinous material that occupies approximately four-fifths of the cavity of the eyeball, behind the lens.
  • the posterior portion of the vitreous is in direct contact with the retina in a region called the “vitreoretinal interface.”
  • the density of the liposome constructs permits targeted delivery of urea to the vitreoretinal interface, and reduces the possibility that the urea will negatively affect other regions of the eye.
  • an “isolated” molecule e.g., an isolated polypeptide or an isolated polynucleotide, is one that is in a form not found in nature, including those which have been purified.
  • an isolated molecule is substantially pure.
  • the term “substantially pure” refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.
  • a “label” is a detectable compound that can be conjugated directly or indirectly to a molecule, so as to generate a “labeled” molecule.
  • the label can be detectable on its own (e.g., radioisotope labels or fluorescent labels) or can catalyze chemical alteration of a substrate compound or composition that is detectable (e.g., an enzymatic label).
  • inhibitor refers to any statistically significant decrease in biological activity, including full blocking of the activity.
  • inhibitor can refer to a decrease of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in biological activity.
  • active agent refers to any substance, other than food, used in the prevention, diagnosis, alleviation, treatment, or cure of a disease.
  • Active agents include protective agents and diagnostic agents.
  • the active agent can include any substance disclosed in at least one of: The Merck Index, 15th Edition (2013); Pei-Show Juo, Concise Dictionary of Biomedicine and Molecular Biology, (2001); U.S. Pharmacopeia Dictionary of USAN & International Drug Names (2014); and Physician's Desk Reference, 70th Edition (2016). See also Stedman's Medical Dictionary, 28th Edition (2013).
  • composition refers to a preparation in which the active agent is in an effective form, i.e., is biologically active and is formulated such that it can be released in an environment and at a concentration that engenders a therapeutic effect, and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered.
  • a composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline.
  • Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. human albumin), a preservative (e.g. benzyl alcohol), an absorption promoter to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.
  • a “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, sports animals, and laboratory animals including, e.g., humans, non-human primates, canines, felines, porcines, bovines, equines, rodents, including rats and mice, rabbits, etc.
  • an “effective amount” of an active agent is an amount sufficient to carry out a specifically stated purpose.
  • An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
  • a subject is successfully “treated” for a disease or disorder of the eye according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder.
  • Prevent refers to prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder.
  • those in need of prevention include those prone to have or susceptible to the disorder.
  • a disease or disorder of the eye is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, than a patient who has not been subject to the methods of the invention.
  • Liposomes that are subunits of the liposome constructs of embodiments of the present invention are SUVs composed of a core enclosed by a bilayer of natural or synthetic origin.
  • the liposomes can comprise one or a mixture of more than one phospholipid.
  • the phospholipids can have different chain lengths, different charges, and can be saturated or unsaturated. Incorporation of cholesterol enhances the stability of liposomes by improving the rigidity of the membrane.
  • the liposomes can utilize cholesterol and lipid-conjugated hydrophilic polymers as the main components. The choice of components and their relative ratios influence the structural stability of the liposomes, their release time, the amount of cargo (i.e., urea) encapsulated, and the process used for encapsulation.
  • the liposome subunits can comprise one or more of: cholesterol, diarachidonoyl phosphatidylcholine (DAPC), dibehenoyl phosphatidylcholine (DBPC), dilauroyl phosphatidylcholine, (DLPC), dimyristoyl phosphatidic acid (DMPA), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), dimyristoyl phosphatidylinositol (DMPI), dimyristoyl phosphatidylserine (DMPS), dioleoyl phosphatidic acid (DOPA), dioleoyl phosphatidylcholine (DOPC), dioleyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), dioleoyl phosphatidylinositol (DOPI), diooy
  • the size of the SUVs is critical.
  • the SUVs preferably have a z-average diameter of between about 50 nm and about 250 nm, preferably between about 140-220 nm, or about 100-200 nm, or about 120-190 nm, or about 150-200 nm, or about 160-180 nm, or about 165-200 nm, as measured by dynamic light scattering.
  • the SUVs have a relatively narrow size distribution.
  • the PdI is between about 0.02 and 0.30. In some embodiments, the PdI is less than about 0.30, 0.25, 0.20, 0.15, 0.125, 0.10, or 0.050.
  • 90% of SUVs in a sample have a diameter of less than about 300 nm, about 270 nm, about 250 nm, or about 220 nm. In some embodiments, 10% of SUVs have a diameter of less than about 120 nm, about 100 nm, about 50 nm, about 20 nm, or about 10 nm, as measured by dynamic light scattering. Preferably, 90%, 95%, or 100% of the SUVs in a sample have a diameter of less than about 250 nm, or less than about 200 nm, or less than about 175 nm, or less than about 150 nm, as measured by dynamic light scattering.
  • the SUVs comprise urea encapsulated within them, i.e., the urea is “cargo” in the central compartment of the SUVs.
  • the SUVs of the various embodiments of the present invention have a specific gravity that is greater than that of vitreous humor.
  • the SUVs have a specific gravity that is greater than about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.15, or about 1.2.
  • Zeta potential measures the electrostatic repulsion between particles of similar charge in a dispersion or solution.
  • the magnitude of the zeta potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles.
  • zeta potential is low, attractive forces between particles can exceed repulsive forces, resulting in more agglomeration.
  • zeta potential is high, the particles resist aggregation.
  • the SUVs have a zeta potential of between about ⁇ 70 mV and about 70 mV, as calculated using electrophoretic light scattering.
  • the zeta potential is less than or equal to zero mV.
  • the zeta potential is 0 ⁇ 5 mV.
  • the zeta potential is about ⁇ 70, ⁇ 65, ⁇ 60, ⁇ 55, ⁇ 50, ⁇ 45, ⁇ 40, ⁇ 35, ⁇ 30, ⁇ 25, ⁇ 20, ⁇ 15, ⁇ 10, ⁇ 5, ⁇ 4, ⁇ 3, ⁇ 2, ⁇ 1, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mV.
  • Zeta potential can be adjusted using methods known in the art, such as by the addition of salts and/or by modifying the pH.
  • the liposome constructs comprise an emulsifier or binding agent to enhance agglomeration.
  • emulsifiers include, without limitation, acacia, glyceryl monooleate, glyceryl monostearate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium lauryl sulfate, sodium dioctyl sulfosuccinate, sorbitan oleate, sorbitan palmitate, sorbitan stearate, and triethanolamine oleate.
  • the SUVs/liposome constructs can comprise a surface modifying group and/or a surface antigen.
  • the surface modifying group is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the level of pegylation of the liposome surface may vary, for example, from 1 mol % to 20 mol %, or higher.
  • the surface antigen is rhodamine.
  • Stability of liposomes depends upon the various properties such as surface charge, size, surface hydration, and fluidity of lipid bilayers.
  • Surface charge determines interaction of liposomes with ocular membrane.
  • the liposomal membrane can have a positive charge, a negative charge, or no (neutral) charge.
  • the individual lipids that comprise the lamella of the SUVs can each have a net positive, a net negative, or a net neutral charge. Local regions of charge can influence the properties of SUVs, even where the net charge is neutral.
  • the liposome constructs can be responsive to stimuli, such as pH, temperature, light, oxidation, enzymatic degradation, radiation, or combinations thereof.
  • the liposome constructs of the embodiments of the present invention can contain at least about 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1 mg urea per ⁇ L of packed liposome construct pellet.
  • Packed liposome construct pellets are prepared by ultracentrifuging a sample containing liposome constructs at about 90,000 g for about 5 minutes and decanting the supernatant.
  • the liposome constructs of the embodiments of the present invention are capable of extending dwell time of urea at the vitreoretinal interface, and can be optimized to release their cargo at the desired rate.
  • the liposome construct e.g., a sustained-release drug delivery system
  • the liposome construct can release urea for at least about 2, 4, 6, 12, 18, 24, 48, or 72 hours, or at least about 1, 2, 3, 4, 5, or 6 weeks after a single administration.
  • the liposome construct can release about 10% or less of the encapsulated urea at 4-8 hours after administration.
  • the liposome construct can release about 50% or less of the encapsulated urea at 8-12 hours after administration.
  • the liposome construct can release at least about 75% of the encapsulated urea at 1 hour after administration. In one embodiment, the liposome construct can release at least about 80% of the encapsulated urea at 8 hours after administration. In one embodiment, the liposome constructs can release at least about 80% of the encapsulated urea at 24 hours after administration. Release rates can be varied depending on the desired dosage by varying the formulation of the liposome constructs.
  • compositions comprising liposome constructs it might be desirable for a composition comprising liposome constructs to have a tiered-release profile.
  • some urea is released immediately after injection, while some urea is released at various time points after injection, e.g., every 6 hours, every 12 hours, every day, every two days, every three days, every week, every month, etc.
  • the composition can comprise a mixed population of SUVs, wherein the SUVs have one or more different properties, such as size, charge, composition of the lipid bilayer(s), modification(s) of the lipid bilayer(s), or a combination thereof, thereby varying the release rate of the urea.
  • the liposome constructs can comprise an antibody or antigen-binding fragment thereof that specifically binds to an antigen expressed in cells of the retina or macula.
  • the cells are Müller cells, retinal ganglion cells, retinal axonal cells, inner limiting membrane cells, retinal pigment epithelial cells, or retinal astrocytes.
  • the antigen is expressed on the surface of the cells. In some embodiments, the antigen is specifically expressed on the surface of the Müller cells.
  • the antigen is selected from the group consisting of vimentin, glutamine synthetase, fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 4 (FGFR4), fibroblast growth factor receptor 9 (FGFR9), Heparin Binding Growth Factor, glial fibrillary acidic protein (GFAP), CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), and retinaldehyde binding
  • SUVs can be made by methods known in the art, such as solvent evaporation, reverse phase evaporation, dehydration-rehydration, detergent dialysis, thin film hydration (Bangham method), detergent depletion, solvent (e.g., ether/ethanol) injection, emulsion methods, dense gas methods, supercritical fluid methods, etc.
  • solvent e.g., ether/ethanol
  • a lipid mixture can be dissolved in an organic solvent and then dried to form a lipid film.
  • the dried lipid film can then be hydrated and sized, for example, by extruding them through orifices of decreasing pore size, which results in liposome constructs comprised of unilamellar liposomes, and having a standardized uniform diameter.
  • the lipid film can be hydrated with a solution of urea, such that it becomes encapsulated within the interior of the SUVs that form liposome constructs.
  • the liposomes can be sized as described above.
  • the urea is in a saturated solution or a supersaturated solution.
  • An alternative method of preparing liposome constructs comprising urea is to load the urea into pre-formed SUVs using a pH gradient method where the aqueous interior of the liposome has a lower pH than the external medium surrounding the liposome construct. Urea will migrate and concentrate within the liposome construct.
  • Another method of loading urea into the interior of liposome constructs employs an ammonium sulfate gradient method.
  • compositions Comprising Liposome Constructs and Methods of Use
  • the SUVs are small enough that they can be sterile-filtered, but can still entrap a therapeutically effective amount of urea. They agglomerate sufficiently upon injection such that they stay together in a liposome construct once administered, rather than dispersing throughout the vitreal fluid, and they are denser than the vitreous, such that they can settle onto and blanket the retinal surface.
  • While “empty” SUVs i.e., without encapsulated urea will agglomerate following intravitreal injection, they disperse readily under gentle agitation of the vitreous humor.
  • the same SUVs with encapsulated urea form a liposome construct that agglomerates following intravitreal injection and does not disperse under gentle agitation of the vitreous humor.
  • Multiple different urea-containing liposome constructs of the invention demonstrate agglomeration that withstands gentle agitation of the vitreous humor, which allows the drug to be released from an agglomerated depot spatially within the vitreous, rather than from a dispersed position throughout the vitreous.
  • the pharmaceutical compositions of the invention provide novel formulations for effectively delivering active agents to the vitreoretinal interface.
  • the liposome constructs of the invention form an agglomeration of SUVs, which agglomeration is stabilized by the encapsulation of urea within the SUVs.
  • the density of the composition causes it to sink in the vitreal fluid, which is optionally facilitated by delivery to a subject in a supine position, resulting in targeted delivery to, and release of urea at, the retinal interface, rather than in the whole eye. Therefore, the disclosure provides a method of increasing the exposure of the retina of a subject to urea, where the method includes administering the liposome construct comprising urea to the eye of the subject.
  • compositions comprising liposome constructs as described above, optionally further comprising one or more carriers, diluents, excipients, or other additives.
  • the compositions can be at a pH of about 5.0 to about 8.5.; preferably, the pH is about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5.
  • Such compositions can include liposome constructs comprising urea.
  • urea can also be present in the carrier or buffer comprising the liposome constructs.
  • concentration of unencapsulated urea in the carrier or buffer can vary depending upon the desired characteristics of the composition.
  • the composition can be formulated to maintain equilibrium between the concentration of encapsulated urea and unencapsulated urea, so that the concentration of urea that is encapsulated remains stable.
  • concentration of unencapsulated urea in the storage carrier or buffer can be different from its concentration in the composition that is administered.
  • the carrier or buffer can comprise a higher concentration of unencapsulated urea if an initial bolus dose is desired upon administration.
  • the composition can be in a variety of forms, such as solution, microparticle, nanoparticle, hydrogel, etc., or a combination thereof.
  • the liposome constructs are dispersed in a gel.
  • the liposome constructs are in the form of an emulsion or a suspension.
  • urea encapsulated in a liposome construct can be administered in a therapeutically effective amount for the in vivo treatment of diseases or disorders of the retina, particularly diabetic retinopathy.
  • the disclosed liposome constructs can be formulated so as to facilitate administration and promote stability of the urea.
  • the liposome constructs of the embodiments of the present invention can be administered in a pharmaceutical composition.
  • compositions in accordance with the present invention can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like.
  • a “therapeutically effective amount” of urea means an amount sufficient to achieve a benefit, e.g., to induce PVD.
  • a suitable pharmaceutical composition can comprise one or more buffers (e.g. acetate, phosphate, citrate), surfactants (e.g. polysorbate), stabilizing agents (e.g. human albumin), and/or salts (e.g., acid addition salts, base addition salts) etc.
  • buffers e.g. acetate, phosphate, citrate
  • surfactants e.g. polysorbate
  • stabilizing agents e.g. human albumin
  • salts e.g., acid addition salts, base addition salts
  • aqueous and non-aqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of a certain particle size in the case of dispersions, and by the use of surfactants.
  • compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, can also be added into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • a pharmaceutical composition provided herein can also include a pharmaceutically acceptable antioxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxy
  • this disclosure provides a method of treating a disease or disorder of the eye, e.g., diabetic retinopathy, wherein the method comprises administering to a subject in need thereof a pharmaceutical composition comprising a liposome construct encapsulating urea, as provided herein, wherein a sufficient amount of the urea reaches and remains in contact with the retina for a period of time sufficient to treat the disease and/or to reach a desired endpoint, such as inducing PVD.
  • the methods of the embodiments of the invention can further comprise positioning the subject in a supine position, i.e., on his or her back, to facilitate migration of the liposome construct composition to the posterior portion of the eye.
  • the liposome constructs are denser than the vitreous, positioning a subject in this manner takes advantage of gravity and causes the liposome constructs to “sink” to the retina, which is located at the back of the eye, thereby achieving targeted delivery to the retina.
  • the herein provided liposome constructs are useful for the treatment or prevention of any disease or disorder that can be addressed by the delivery of urea to the retina, including the macula.
  • diseases or disorders that can be treated or prevented using the liposome constructs and methods of the embodiments of the invention include one or more of age-related macular degeneration (AMD), branch or central retinal vein occlusion, central serous chorioretinopathy, choroidal detachment, congenital X-linked reinoschisis, diabetic macular edema (DME), diabetic retinopathy (DR), epiretinal membranes, familial exudative vitreoretinopathy, infectious retinitis, macular edema, macular hole, macular pucker, persistent fetal vasculature, presumed ocular histoplasmosis syndrome, retained lens fragment, retinoblastoma, retinal tears or detachment, retinitis pigmentosa, retinopathy of prem
  • Clinical response to administration of a liposome construct can be assessed using standard screening techniques, for example, optical coherence tomography (OCT), fundus photography, or fluorescein angiography. Clinical response can also be assessed by improvement in the symptoms associated with the disease or disorder.
  • OCT optical coherence tomography
  • the targeting of liposome constructs can be analyzed by observing fluorescent markers on or in the liposome constructs.
  • administration can be via intravitreal injection, intravitreal implantation, iontophoresis, or a microelectromechanical device.
  • administration is via intravitreal injection, such as, for example, via an 18-31 gauge needle.
  • administration is via a 27-gauge needle or a 30-gauge needle.
  • the volume that is typically delivered via vitreal injection is between about 50 ⁇ L and about 150 ⁇ L, preferably about 100 ⁇ L.
  • the concentration of liposome constructs of the embodiments of the invention that can be combined with carrier materials to produce a dosage form will vary depending upon many different factors, including the encapsulation efficiency of urea, whether treatment is prophylactic or therapeutic, other medications administered, and whether the patient is human or an animal.
  • the amount of liposome construct to be administered is readily determined by one of ordinary skill in the art without undue experimentation, given this disclosure. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
  • composition comprising liposome constructs can be administered as a single dose or multiple doses.
  • the composition can be administered as many times as needed to achieve a targeted endpoint, such as PVD induction. Injection intervals may vary.
  • the composition can be administered every 6, 12, 24, 48, or 72 hours, every 1, 2, 3, or 4 weeks, or every 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36, or 48 months. Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).
  • compositions can also be administered in combination therapy and/or combined with other agents.
  • This disclosure provides for the use of a pharmaceutical composition comprising a liposome construct encapsulating urea, as described herein, to treat or prevent diseases or disorders of the retina or macula.
  • This disclosure also provides for the use of liposome constructs comprising urea as described herein in the manufacture of a medicament for treating or preventing diseases or disorders of the retina or macula.
  • the disclosure further encompasses the use of a pharmaceutical composition comprising a liposome construct comprising urea for prevention, management, treatment, or amelioration of one or more symptoms associated with disease, disorder, or injury of the eye, either alone or in combination with other therapies.
  • kits comprising liposome constructs and/or compositions as provided herein and instructions for use.
  • the kit can further contain at least one additional reagent, or one or more additional liposome constructs.
  • Kits typically include a label indicating the intended use of the contents of the kit.
  • label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • kits that comprise one or more liposome constructs, which can be used to perform the methods described herein.
  • a kit comprises at least one type of liposome construct of the invention in one or more containers.
  • the kits contain all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.
  • the disclosed liposome constructs can be readily incorporated into one of the established kit formats which are well known in the art.
  • Embodiments of the present invention can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain liposome constructs of the present invention and methods for using liposome constructs of the present invention. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.
  • Formulation Rationale 1 58 mol % DPPC Original formulation 42 mol % cholesterol DPPC: saturated High cholesterol affects liposome stability in vitreal fluid 2 58 mol % DOPC Substitute saturated DPPC with unsaturated DOPC (two 42 mol % cholesterol chains) to evaluate a more “leaky” liposome 3 58 mol % POPC Substitute DPPC with more biosimilar POPC (one 42 mol % cholesterol unsaturated chain and one saturated chain) 4 53 mol % DPPC Replace 5 mol % DPPC with DPPE-PEG2000 to assess 42 mol % cholesterol whether a PEG coating affects stability 5 mol % DPPE-PEG2000 5 29 mol % DPPC Replace half of the DPPC with DPPG to evaluate the effect 42 mol % cholesterol of an overall negative charge on the liposomes 29 mol % DPPG 6 80 mol % POPC Use POPC/DOTAP to evaluate the
  • lipids were obtained from Avanti Polar Lipids, Inc. (Alabaster, Ala.). Formulations 1, 3, 8, 11, and 12 were prepared with carboxyfluorescein. Briefly, a 100 mM carboxyfluorescein solution was prepared in 1 ⁇ PBS (HyCloneTM Cat. No. SH0256, GE Healthcare, Marlborough, Mass.), and the pH was adjusted to 6.5-7.5 with 1% NaOH. This solution was filtered and used to prepare self-quenching liposomes. Lipid films were dried under a stream of nitrogen, followed by vacuum for a minimum of 2 hours, and were rehydrated with the 100 mM carboxyfluorescein solution.
  • PBS HyCloneTM Cat. No. SH0256, GE Healthcare, Marlborough, Mass.
  • the rehydrated liposomes were extruded through a 0.2 ⁇ m filter membrane (Millex®, MilliporeSigma, Darmstadt, Germany).
  • the liposomes were separated from the unencapsulated carboxyfluorescein by size-exclusion chromatography on a Sephadex G75 column (Sigma Aldrich, St. Louis, Mo.).
  • the plate was sealed with a self-adhesive plastic film and shaken vigorously for 3 cycles in a VMax Kinetic Microplate Reader (Molecular Devices, Sunnyvale, Calif.) before fluorescence emission was determined.
  • the gain was set at 800, as determined from the gain required to produce 50% maximum emission in a well containing liposomes with PBS and RIPA buffer at Time 0.
  • PVD posterior vitreous detachment
  • Formulations 1, 2, 3, 12, and 14 were prepared. All lipids except cholesterol were obtained in chloroform solution; cholesterol powder was added to the chloroform solution in the desired ratio. Cholesterol was evaporated via a nitrogen stream, followed by freeze-dry evaporation of chloroform-lipid samples. The resulting lipid cake was hydrated with 100 ⁇ L of 1 g/mL urea (Invitrogen Cat. No. 15505035, Carlsbad, Calif.) solution, agitated for 30 min. at 4° C., and extruded using a two-step extrusion process with 0.8 ⁇ m and 0.2 ⁇ m filters.
  • 1 g/mL urea Invitrogen Cat. No. 15505035, Carlsbad, Calif.
  • the amount of encapsulated urea and the urea loading (encapsulation) efficiency was determined for each formulation. Following 0.2 ⁇ m filter extrusion, liposome samples were ultra-centrifuged (Airfuge®, Beckman Coulter, Indianapolis, Ind.) at 90,000 g for 5 minutes at room temperature to separate free urea in solution from the encapsulated urea in the packed liposome pellet. The supernatant was decanted. The upper limit for encapsulation efficiency was found by measuring the amount of urea encapsulated within the packed pellet mass (re-suspended in 100 ⁇ L DI H 2 O) and dividing by the mass of urea in the loading buffer.
  • the packed pellet was washed a second time with 100 ⁇ L of PBS in order to remove any urea associated with the liposome particles but not truly encapsulated.
  • the amount of urea encapsulated within the washed packed pellet mass (re-suspended in 100 ⁇ L DI H 2 O) divided by the mass of urea in the loading buffer provides the lower limit of the encapsulation efficiency.
  • the lower limits of loading efficiency and total mass of encapsulated urea are shown in Table 3.
  • the upper limits of loading efficiency and total mass of encapsulated urea are shown in Table 4.
  • Particle size analysis was performed using a Microtrac (Montgomeryville, Pa.) 150 instrument and Microtrac Particle Size Analyzer software, version 10.1.3.
  • Particle size analysis of Formulation 2 without urea showed that 90% of liposome constructs were under 250 nm, and that 10% were under 100 nm.
  • the liposome construct particle size of Formulation 2 without urea ranged from about 100 nm to about 250 nm.
  • Particle size analysis of Formulation 2 with encapsulated urea showed that 90% of liposome constructs were under 300 nm and that 10% were under 90 nm.
  • the liposome construct particle size of Formulation 2 with encapsulated urea ranged from about 90 nm to about 300 nm.
  • Optimal buffer compositions were assessed for encapsulation efficiency and stability of the liposome constructs at 4° C. for 96 hours.
  • Formulation 2 liposome constructs with encapsulated urea were made with 6 different buffer compositions, shown in Table 5.
  • Buffer Formulation 1 0.95 g/mL urea in diH2O 2 0.95 g/mL urea in 0.5x PBS 3 0.95 g/mL urea in 1x PBS 4 0.95 g/mL urea in 2x PBS 5 0.95 g/mL urea + citric acid (pH 6.5) 6 0.95 g/mL urea + citric acid (pH 6.5) + 10% sucrose
  • Each buffer formulation is an aqueous hydration medium that was added to dry lipid cake (Formulation 2), followed by extrusion, as described above.
  • the loading efficiency and total mass of encapsulated urea are shown in Table 6.
  • the buffer composition of deionized water and urea had the highest urea encapsulation efficiency; 0.5 ⁇ PBS and 1 ⁇ PBS buffers with urea were the next best.
  • stability of liposome constructs in the buffer containing citric acid (pH 6.5), 10% sucrose, and 0.95 g/mL urea did not demonstrate a change in encapsulated urea after 96 hours.
  • the 2 ⁇ PBS urea buffer showed the least stability, with an encapsulated urea concentration loss of roughly 30%.
  • Formulation 2 liposome constructs were used to evaluate liposome construct behavior with higher volume-to-volume concentrations of liposome constructs.
  • pellets were prepared as described above, two pellets were combined, re-suspended to a total volume of 100 ⁇ L 0.5 ⁇ PBS, and aspirated through a 27-gauge or 30-gauge needle.
  • the amounts of encapsulated and free urea were determined as described above, and are shown in Table 7.
  • the volume of aqueous portion of a 100 ⁇ L sample was separated from the pellet volume via centrifugal filtration, measured, and the pellet volume calculated.
  • the total urea and the free urea concentrations were measured by high-performance liquid chromatography (HPLC) and used to calculate the encapsulated urea concentration and encapsulation efficiency (degree of urea incorporation).
  • Zeta potential and particle size and distribution were measured by laser light scattering (LLS) using a Zetasizer (Malvern Instruments Ltd., Worcestershire, UK). Injectability through a 30G needle was confirmed by testing the total and free urea concentrations, and calculating the encapsulated urea concentration again.
  • the volume lost during filtration through a 0.2 ⁇ m filter was ascertained by measuring the volume of a sample before and after filtration.
  • the specific gravity of the batch was measured gravimetrically for a known volume, and agglomeration in vitreous humor or PBS was visually evaluated.
  • the in vitro release of encapsulated urea was measured using a 20 kDa dialysis cassette at 37° C., over 7 days, with a sample volume:buffer volume ratio of 100 ⁇ L:5 mL.
  • the total urea concentration was measured by HPLC, and the amount of encapsulated urea released was calculated based on the initial free urea concentration and the total urea concentration at each respective time point.
  • the composition of Batches F5 and F5B was 58 mol % DOPC and 42 mol % cholesterol; the batch size was 17 grams (1500-1600 doses). A 600 mg/mL saturated urea solution was used to make urea-encapsulated liposome constructs. After micro-fluidization, the batch was homogenized further to reduce the particle size of the liposomes to an acceptable size.
  • the batch was aliquoted into two volumes, which were separately concentrated to a 40% liposome:60% saturated urea buffer (vol/vol ratio), using a stir cell device (Amicon, 50 mL, max pressure 75 psi) with a 10 kDa cut-off ultrafiltration membrane (MilliporeSigma, Darmstadt, Germany, Cat. No. PLGC04310), and sterile filtered.
  • the final volume of batch F5 was 105 mL, and the final volume of batch F5B was 51 mL.
  • Batches F11-F13 also had a composition of 58 mol % DOPC and 42 mol % cholesterol.
  • the composition of Batch F6 was 91 mol % DOPC and 9 mol % cholesterol; the batch size was 3 grams (1500-1600 doses). A 450 mg/mL saturated urea solution was used to make urea-encapsulated liposome constructs. The batch was concentrated and sterile filtered as described for Batches F5 and F5B.
  • the composition of Batch F8 was 70 mol % DOPC and 30 mol % cholesterol; the batch size was 3 grams (1500-1600 doses). A 450 mg/mL saturated urea solution was used to make urea encapsulated liposome constructs. The batch was concentrated and sterile filtered as described for Batches F5 and F5B.
  • the composition of Batch F9 was 45 mol % DOPC and 55 mol % cholesterol; the batch size was 3 grams (1500-1600 doses).
  • a 450 mg/mL saturated urea solution was used to make urea-encapsulated liposome constructs.
  • the batch was microfluidized for 20 minutes, with particle size of the batch measured every 3 minutes. After 20 minutes of microfluidization, the liposome particle size reached 1200 nm, well above the 200 nm target. Phase separation was observed when the sample volume was stored overnight after microfluidization. Homogenization of the batch was performed with the beadbeater for 5 minutes in order to successfully pass the sample through a 0.8 ⁇ m filter.
  • the batch was concentrated and sterile filtered as described for Batches F5 and F5B. Sterile filtration through a 0.2 ⁇ m filter was very difficult, and at least 1 ⁇ 3 of the batch volume was lost during processing, due to complications from the high amount of large particles.
  • the composition of Batch F10 was 58 mol % DOPC and 42 mol % cholesterol, with approximately 6% (w/w) of the cholesterol having a fluorescent label.
  • the batch was made by reverse phase evaporation, followed by microfluization, sonication, and filtration, as described above.
  • Results of characterization testing are shown in Table 10. Particle size and distribution are shown in Table 11. In vitro release data are shown in Table 12, Table 13, and FIG. 5 .
  • This Example provides an evaluation of the tolerability and toxicity of encapsulated urea after intravitreal (IVT) injection into the eyes of New Zealand white rabbits, as well as its efficacy for inducing PVD, and the settling pattern of intravitreally injected liposome constructs encapsulating urea.
  • IVT intravitreal
  • Female New Zealand white rabbits were obtained from Western Oregon Rabbit Co. (Philomath, Oreg.) and were housed and cared for in compliance with the regulations of the USDA Animal Welfare Act and under the review and approval of the institution's Animal Care and Use Committee.
  • Group 1 (Subgroups 1a, 1b, 1c): Acute/Urea Encapsulated Liposomes
  • urea-encapsulated liposome constructs (58 mol % DOPC, 42 mol % cholesterol) intravitreally (IVT) as a single dose into both eyes (OU).
  • Animals were anesthetized with an intramuscular (IM) injection of ketamine hydrochloride (30 mg/kg), xylazine (5 mg/kg), and acepromazine (3 mg/kg) followed by isoflurane by inhalation (1-2.5%) in oxygen (1 L/min).
  • One to two drops of topical proparacaine hydrochloride anesthetic (0.5%) were applied to the animal's eyes prior to the surgical procedure. Animals were kept anesthetized with their heads stabilized for three hours post dose, with one eye facing up and the other eye facing down.
  • VH Vitreous humor
  • Group 2 (Subgroups 2a, 2b): Chronic/Free Urea
  • Two additional animals were administered a single or double dose of free urea solution IVT into the right eye (OD) and balanced salt solution (BSS) into the left eye (OS).
  • Animals were anesthetized with an IM injection of ketamine hydrochloride (30 mg/kg) and xylazine (5 mg/kg).
  • One to two drops of topical proparacaine hydrochloride anesthetic (0.5%) were applied to the animal's eyes prior to the surgical procedure.
  • OCT optical coherence tomography
  • PVD posterior vitreous detachment
  • OCT imaging revealed additional instances of PVD that were not revealed by clinical ophthalmic examinations. It can be concluded that PVD developed in the eyes of both animals treated with free urea solution within 2 days of dosing and persisted for the rest of the study, even if ophthalmic examinations and imaging did not always detect it. Because Group 1 animals did not undergo OCT imaging, instances of PVD after treatment with urea-encapsulated liposome constructs may have been undetected.
  • Subgroup 2a and Subgroup 2b animals exhibited clouding of the OD vitreous from Day 0 through Day 3. No such clouding was observed in the OS vitreous of either subgroup. The clouding was most likely an effect of vitreal protein changes due to the urea, as the cloudiness was not seen in the vehicle-treated eyes. Additionally, the cloudiness was likely not due to infection, as the vitreous in the urea-treated eyes began to clear after 3 days post dose.
  • OCT imaging showed PVD in the right eye (OD) of the Subgroup 2a animal on Day 3.
  • OD right eye
  • AM and PM Days 4
  • No PVD was seen in in these animals OD at other imaging time points.
  • Neither animal exhibited PVD in the left eye (OS) at any time point.
  • OCT images also showed that the regions of retinal folding seen in the fundus imaging were areas where the retina had detached mostly at the ganglion cell layer and, in fewer cases, at the inner nuclear cell layer.
  • the retinal detachment peaked between Days 3 and 7 and then began to subside, as reflected by retina re-attaching.
  • Substantial numbers of hyper-fluorescent cells were observed in the retina after re-attachment, most likely indicating immune cell infiltration.
  • FIG. 8A-8B Representative images are shown in FIG. 8A-8B .
  • This Example provides an evaluation of the tolerability of reduced (compared to Example 5) concentrations of free urea after IVT injection into the eyes of New Zealand White rabbits (non-GLP), and a determination of the time it takes to induce a PVD after injection of each of the various dose strengths.
  • each animal Prior to placement in the study, each animal underwent an ophthalmic examination (slit-lamp biomicroscopy and indirect ophthalmoscopy). Ocular findings were scored according to a modified McDonald-Shadduck Scoring System. The acceptance criteria for placement on study were scores of “0” for all variables.
  • Ophthalmic examinations (slit-lamp biomicroscopy and indirect ophthalmoscopy) were performed on Days ⁇ 3 or 0 (baseline prior to test/control article administration), 1, 4, 7 or 8, 14, 21, and 35.
  • Day 4 examinations were performed at the beginning of the workday.
  • Day 21 examinations were performed only on Groups 1, 2, and 5.
  • Ocular findings were scored according to a modified McDonald-Shadduck Scoring System. All animals had no ocular anomalies during the baseline pre-screening examination. Observations included assessment of the development and time course of PVD.
  • Dilated choroidal and/or retinal vessels were observed in all animals in one or both eyes at some or all clinical ophthalmic examination time points up to 21 days after administration. The dilatation was generally limited to the region of injection. As this finding was sometimes noted in both eyes, including the left eye (OS) injected with BSS only, it was likely a reaction to the IVT injection procedure, rather than an effect of the urea.
  • OS left eye
  • a ring-shaped opacity around the posterior lens capsule in the Group 4 (50 mg/eye urea) animal seen in the urea-treated right eye (OD) on the day after test article administration may have been due to irritation of the lens tissue by the high concentration of urea.
  • PVD was difficult to visualize via clinical ophthalmic examinations; however, OCT imaging was able to identify cases of PVD (see below).
  • Images of the fundus were taken on Days ⁇ 3 or 0 (baseline prior to drug or BSS administration), 1, 4, 7 or 8, 14, 21, and 35. Day 4 images were taken at the beginning of the workday. Day 21 images were taken only of Groups 1, 2, and 5. Animals were not anesthetized for imaging.
  • OCT OCT was performed on Days ⁇ 3 or 0 (baseline prior to test/control article administration), 1, 4, 7 or 8, 14, 21, and 35.
  • Day 4 images were taken at the beginning of the workday.
  • Day 21 images were taken only of Groups 1, 2, and 5. Animals were anesthetized as described above. A total of 8 images per rabbit per day was acquired.
  • Retinal degradation, subretinal fluid, and signs of retinitis were observed in the OCT images of Group 3 and 4 animals starting the day after urea administration and in the Group 2 animal seven days after urea administration.
  • OCT imaging revealed PVD in the treated eyes (OD), as described in Table 18. PVD was observed as a partial line of cellular components in the vitreous cavity with an underlying dark/black area with acellular components. Representative images are shown in FIG. 10A-10B .
  • B-Scan ultrasound images were taken on Days ⁇ 3 or 0 (baseline prior to test/control article administration) and on Day 35.
  • Day 35 B-scan images of the Group 4 animal showed evidence of PVD in the right eye (OD), but not the left eye (OS).
  • OS right eye
  • B-scan images of other animals no PVD could be detected. Representative images are shown in FIG. 11A-11B .
  • This Example provides observation of the physical location of liposome constructs in the posterior ocular space immediately after intravitreal injection of a wide range of dose concentrations and to evaluate the local effects of the drug following treatment.
  • Liposomes were Formulation 2 (58 mol % DOPC, 42 mol % cholesterol). The study design is summarized in Table 19. Each group had an N of 1.
  • OCT scans were taken via Bioptigen Envisu, high resolution fundus images were captured of the drug product location via the MicronX and a digital image of the dosed eyes was taken with an iPhone over the course of 32 days. Baseline images for all animals were obtained via OCT, MicronX and the digital camera. For Groups 1-4, treatment of the OS eye only allowed for animals to remain on their side with their eye facing upward and allowing the drug product to settle to the back of the eye.
  • PVD was observed in every treated eye with quicker response times with the higher concentration doses.
  • the presence of particles (cloudiness) inside the vitreous chamber throughout the 4 weeks of study was generally confirmed by photography, retinal imaging, and OCT.
  • OCT optical coherence tomography

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